Since the 1980's when the human immunodeficiency virus (HIV) was discovered, 30 million people have died, making HIV the 6th leading cause of death in the world. If untreated, HIV infection eventually causes acquired immune deficiency syndrome (AIDS) a serious insult to the human immune system. So far, the treatments of choice for HIV/AIDS are antiretroviral drug therapies, but they are treatments rather than cures in that the HIV virus still remains in the body. Work on developing effective therapies that suppress the replication of HIV and hence cure the disease is ongoing. Interruption in any one of the steps in the HIV life cycle has the possibility to stop replication, the process by which viruses use the host cell to make new copies of themselves. A promising target is tRNALys3, the primer of reverse transcriptase that is recruited by the HIV-1 virus during virus RNA replication. Different from other tRNA, tRNALys3 has chemically-rich posttranscriptional modifications in the anticodon stem and loop (ASL) domain—one is 5-methylmethoxymethyl-2-thiouridine (mcm5s2U34) at position 34, and another 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A37) at position 37. Blocking the recruitment of tRNALys3 has the potential to interfere with the HIV life cycle, causing the death of the virus.
A variety of candidate peptide sequences that mimic the binding behavior of nucleocapside proteins in the body were synthesized and then tested for their capability to bind the anticodon stem and loop (ASL) of tRNALys3. Twenty different peptide sequences containing 15 or 16 amino acids were chosen from Peptide Phage Display Libraries and fluorescence and circular dichroism spectroscopy was used to characterize the peptide binding to these ASLs. The best peptide sequence—RGVFSHPHTAVPSHN (SEQ ID NO:1) exhibited a relatively high binding affinity for hypermodified ASLLys3, but bound poorly to singly modified ASLLys3, the ASLs of the two other human tRNALys species, AsLLys1, 2 and Escherichia coli ASLGlu and ASLVal.
Other research groups have also investigated the binding behavior of RNA and proteins. Xia et al. used a combination of fluorescence up-conversion and transient absorption techniques to study the mechanisms and dynamical processes associated with RNA-protein recognition. They found that the complex formed by the antiterminator N protein and the stem-loop RNA hairpin exists in a dynamical two-state equilibrium between stacked and unstacked conformations. Formation of the stacked structure was driven by hydrophobic interactions (rather than by charge-charge interactions) between the residue at site 14 of their peptide chain and the ribose on RNA. In related work, Zhang et al. utilized site-directed spin labeling to examine the distribution of conformations at the interface between a peptide of 22 amino acids and a stem-loop RNA element. They observed that the C-terminal fragment of the bound peptide tends to adopt multiple discrete conformations within the complex.